Biochemistry, Department of


Date of this Version



Biochemistry. 2011 July 26; 50(29): 6508–6517.


Copyright © 2011 American Chemical Society. Used by permission.


Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises

production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly

reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases,

impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a

catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step

in glutathione production. Four clinical missense mutations have been identified within GCLC:

Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of

these mutations on enzymatic function in vivo and in vitro to gain further insights into the

pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wildtype

or mutant GCLC and cellular glutathione levels were determined. The four mutant

transfectants each had significantly lower levels of glutathione relative to wild-type, with the

Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL

activity were investigated using an S. cerevisiae model system. Mutant GCLC alone could not

complement a glutathione-deficient strain and required the concurrent addition of GCLM to

restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the

Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can

largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic

constants comparable to wild-type GCLC, suggesting that heterodimer formation is needed for

stability in vivo. Strategies that promote heterodimer formation and persistence would be effective

therapeutics for the treatment of GCL deficiency.